This proposal investigates how co-evolution of retrotransposon elements (RTEs) and KRAB zinc finger proteins (KZNFs) have resulted in new gene regulatory modules and how these modules contribute to human embryonic development and pluripotency. KZNFs are the largest family of human transcription factors and have undergone rapid evolution in primates to repress RTE expression. Both RTEs and KZNFs are highly expressed in pluripotent stem cells (PSCs) and are aberrantly expressed in cancers and neurological diseases, suggesting that they play functional roles in these cell types. KZNFs show strong signals of selection and activity even after their target RTEs have lost the ability to mobilize (i.e. generate new insertion events) and therefore no longer pose a threat to their host genome. We hypothesize that over evolutionary time KZNFs are maintained by the host to regulate host gene expression after their job repressing RTEs is finished, and hence tracing the evolution of KZNF-RTE and KZNF-enhancer interactions will not only increase our understanding of the rules governing ZNF-DNA binding, but open a new window on the evolution of and mechanisms in primate/human gene regulatory networks. We will use comparative genomics approaches that take advantage of new, highly contiguous, primate genome assemblies to trace this evolutionary history. This analysis will allow us to apply assays we have developed to dissect the role of evolutionary changes to both KZNFs and their RTE/host targets in controlling transcription in PSCs, and by analogy, in early embryonic cell types. We will focus our experimental analysis on ?naive? and ?primed? PSCs, which mimic epiblast cells of pre- and post-implantation embryos, respectively. These cell types show high expression of a number of specific RTEs and KZNFs, and these expression signatures differ between closely related species, including between human and non-human apes. Naive and primed PSCs are experimentally tractable and critical for regenerative medicine efforts. We have succeeded in making them in human, chimpanzee and orangutan. We will test the function of KZNF-RTE interactions active in these ape PSCs by modulating both KZNF and RTE expression and will assess the consequences of these manipulations on cell fate specification, maintenance of pluripotency and differentiation potential. By performing these experiments in non-human ape PSCs in addition to human PSCs we can identify conserved and species-specific regulatory programs and dissect the molecular and evolutionary basis for recently evolved differences in pluripotency in humans. The results of this work will reveal how RTEs and KZNFs have influenced human evolution and development and provide important insights into the establishment and maintenance of pluripotent stem cells. ? ?